EP2678683B1 - Méthode permettant de pronostiquer l'évolution de l'infection par le vih - Google Patents

Méthode permettant de pronostiquer l'évolution de l'infection par le vih Download PDF

Info

Publication number
EP2678683B1
EP2678683B1 EP12707649.5A EP12707649A EP2678683B1 EP 2678683 B1 EP2678683 B1 EP 2678683B1 EP 12707649 A EP12707649 A EP 12707649A EP 2678683 B1 EP2678683 B1 EP 2678683B1
Authority
EP
European Patent Office
Prior art keywords
hiv
antibodies
prognosis
marker
disease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Not-in-force
Application number
EP12707649.5A
Other languages
German (de)
English (en)
Other versions
EP2678683A1 (fr
Inventor
Laurence MEYER
Vincent Vieillard
Patrice Debre
Joël Crouzet
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut National de la Sante et de la Recherche Medicale INSERM
INNAVIRVAX
Original Assignee
Institut National de la Sante et de la Recherche Medicale INSERM
INNAVIRVAX
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institut National de la Sante et de la Recherche Medicale INSERM, INNAVIRVAX filed Critical Institut National de la Sante et de la Recherche Medicale INSERM
Priority to PL12707649T priority Critical patent/PL2678683T3/pl
Priority to EP12707649.5A priority patent/EP2678683B1/fr
Priority to RS20160990A priority patent/RS55354B1/sr
Publication of EP2678683A1 publication Critical patent/EP2678683A1/fr
Application granted granted Critical
Publication of EP2678683B1 publication Critical patent/EP2678683B1/fr
Not-in-force legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV

Definitions

  • the present invention relates to the field of prognosis of the HIV disease.
  • HIV ELISA test kits use polystyrene microwell strips which are pre-coated with a mixture of HIV antigens, which HIV antigens may consist of recombinant HIV antigens expressed in E. coli, such as recombinant HIV-1 gp41, gp120 and HIV-2 gp36 glycoproteins.
  • ART anti-retroviral pharmaceutical treatment
  • HIV disease staging and classification systems are critical tools providing clinicians and patients essential information for clinical management.
  • Two major classification systems currently are in use, respectively (i) system the World Health Organization (WHO) Clinical Staging end Disease Classification System and (ii) the US Centers for Disease Control and Prevention (CDC) classification.
  • WHO World Health Organization
  • CDC US Centers for Disease Control and Prevention
  • the WHO Clinical Staging and Disease Classification System of HIV/AIDS is mainly based on the determination of the occurrence of clinical events without compulsory requiring the results from laboratory testing.
  • the WHO clinical staging system has been widely used in resource-limited countries and has proved pragmatic and useful in facilities at both the first level and the referral level.
  • the CDC disease staging system assesses the severity of the HIV disease by CD4+ T lymphocyte cell (CD4) counts and by the presence of specific HIV-related conditions.
  • CD4+ T lymphocyte cell (CD4) counts includes all HIV-infected individuals with CD4 counts of less than 200 cells/ ⁇ l, or a CD4 percentage (over all lymphocytes) of less than 14%, as well as those with certain HIV-related conditions and symptoms.
  • the CD4 count consists of the standard laboratory test for assessing HIV stage and prognosis, and for monitoring progression to AIDS and risk of opportunistic illnesses.
  • the CD4 cell count also guides the physician in formulating differential diagnoses in symptomatic patients, and in deciding about initiating antiretroviral treatment (ART), and beginning prophylaxis for opportunistic infections.
  • ART antiretroviral treatment
  • HIV replication In untreated HIV infection, HIV replication usually produces billions of new HIV copies daily.
  • Plasma HIV RNA (viral load) testing quantifies the HIV viral burden in the plasma.
  • the viral load In areas with access to viral load monitoring, the viral load is a standard tool used to monitor treatment response in patients taking ART and, in conjunction with the CD4 cell count, to assess HIV progression (see notably Mellors et al., 1996, Science, Vol. 272(5265): 1167-170 ).
  • AIDS is a pandemic disease affecting more than 30 million people throughout the world with more than 2 millions new infected people per year. To date, AIDS remains a fatal disease which has killed more than 2 million people in 2009.
  • the present invention relates to an in vitro method for the prognosis of progression of an HIV-1 disease in a patient infected with an HIV-1 virus, who has not the rare phenotype of asymptomatic long-term survival, which prognosis is based on the level of antibodies directed against 3S peptide of SEQ ID NO. 2 as a prognostic marker, which prognostic marker is independent from the CD4 count prognosis marker, the said method comprising the steps of :
  • the present invention provides for a novel independent marker that is indicative of the progression status of an HIV-1 disease in HIV-1-infected patients.
  • the present invention provides for novel methods for determining the prognosis of progression of an HIV-1 disease, using the said novel independent marker.
  • the level of antibodies directed against a specific HIV-1-derived peptide is indicative of the status of progression of the HIV-1 disease of the said individual.
  • the said 3S-peptide which consists of a peptide that is derived from the gp41 glycoprotein from an HIV-1 virus, is known per se.
  • the 3S peptide has been shown in the art to bind to gC1qR on CD4+ T cells and to induce the membrane expression of the NKp44L protein in CD4 T cells ( Fauster Bovendo et al., 2010, PLOS Pathogens, Vol. 6(7 ); and WO 2010/040853 ).
  • NKp44L expression at the surface of the CD4 T cells plays a key role in the lysis of CD4 T cells by activated NK cells during HIV-1-1 infection and consequently that specific antibodies directed against the 3S-peptide could affect disease course (see Vieillard et al., 2005, Proc. Natl. Acad. Sci. USA, Vol. 102 : 10981-10986 ).
  • ALT HIV-1-infected patients belonging to the very specific patients cohort
  • ALT which cohort comprises exclusively HIV-1-infected patients having the rare phenotype of asymptomatic long-term survival
  • Vieillard et al.(2006) have shown the existence, in the said ALT cohort of patients, of a correlation between (i) a decrease in the number of CD4 + T cells and (ii) a decrease in the anti-3S antibody level.
  • Vieillard et al.(2006) concluded that the presence of anti-3S antibodies might affect disease course in inhibiting NKp44L expression and CD4 sensitivity to NK lysis. Also, Vieillard et al. (2006) stated that the presence of anti-3S antibodies in the serum helped to control the CD4 cell count. These authors observed that depletion of the overall CD4 T-cell population was closely related to the disappearance of anti-3S antibodies. These authors also added that the production of anti-3S antibodies could possibly contribute to slowing down the progressive depletion of CD4 T cells.
  • the level of anti-3S antibodies consists of a reliable marker of the progression status of the HIV-1 disease in a HIV-1-infected patient.
  • the inventors have shown that the level of anti-3S antibodies is reliably indicative of the HIV-1 disease progression.
  • the anti-3S antibody level consists of a marker indicative of the HIV-1 disease progression, even when the said marker is used alone.
  • the anti-3S antibody level consists of a HIV-1 disease progression marker that is independent from other conventionally used HIV-1 prognosis markers, including the CD4 count prognosis marker and the viral load prognosis marker.
  • results according to the present invention have been obtained from a general cohort of HIV1-infected patients, namely a cohort of HIV-1 seropositive individuals, and not from a cohort of patients having the rare phenotype of asymptomatic long-term survival like the ALT cohort studied by Vieillard et al. (2006).
  • the invention provides a method for prognosis of the severity of the HIV-1 disease progression, which method may be used notably for monitoring the course of AIDS, as well as for monitoring the therapeutic efficiency of anti-retroviral treatments.
  • the present invention relates to an in vitro method for the prognosis of progression of an HIV-1 disease in a patient infected with an HIV-1 virus who has not the rare phenotype of asymptomatic long-term survival, which prognosis is based on the level of antibodies directed against 3S peptide of SEQ ID NO. 2 as a prognostic marker, which prognostic marker, the said method comprising the steps of:
  • HIV-1 disease encompasses basically all the physiological conditions that are undergone by an individual who has been infected by a HIV-1 virus, starting from the time of the virus infection event until the date of the individual's death, irrespective of whether the individual's death is a direct or indirect consequence of the virus infection event. It is recalled that the infection of an individual with a HIV-1 virus causes a chronic disease state that progressively causes a reduction of the effectiveness of the immune system and leaves the HIV-1-infected individuals susceptible to opportunistic infections and tumors.
  • a HIV-1 disease encompasses the primary infection (or acute infection) time period, the seroconversion time period, the asymptomatic stage time period, the early- and medium-stage of HIV-1 symptomatic disease, as well as the late stage of HIV-1 disease (also called AIDS).
  • anti-3S antibodies that are directed against the peptide of SEQ ID N° 2 encompass antibodies that bind specifically to a reference polypeptide having an amino acid sequence comprising the peptide sequence NH 2 -SWSNKS-COOH (SEQ ID N° 1). These antibodies are collectively termed "anti-3S antibodies" in the present description. It may also corresponds to antibodies binding to a peptide having an amino acid sequence that is closely related to sequence of SEQ ID N° 2.
  • Peptides having a sequence closely related to SEQ ID N° 2 encompass peptides comprising an amino acid sequence selected from the group consisting of SEQ ID N° 3 and SEQ ID N°4, which includes peptides having 16 amino acid residues in length and comprising an amino acid sequence selected from the group consisting of SEQ ID N° 3 and SEQ ID N°4, e.g. which includes (i) a variant of the peptide of SEQ ID N°2 wherein the amino acid residue Serine at position 11 is replaced by a threonine residue and (ii) a variant of the peptide of SEQ ID N°2 wherein the amino acid residue Lysine at position 10 is replaced by an arginine residue.
  • antibodies directed against a 3S-peptide encompass antibodies that bind to the peptide of SEQ ID N° 2.
  • the anti-3S antibodies preferably consists of polyclonal antibodies that bind to a 3S peptide, and which are contained in a sample collected from a HIV-infected individual.
  • the 3S-peptide consists of the peptide consisting of the amino acid sequence of SEQ ID N° 2.
  • the "level" of a HIV disease prognosis marker consists of a quantitative value of the said prognosis marker in a sample, e.g. in a sample collected from an HIV-infected patient.
  • the said quantitative value does not consist of an absolute value that is actually measured, but rather consists of a final value resulting from the taking into consideration of a signal to noise ratio occurring with the assay format used, and/or the taking into consideration of calibration reference values that are used to increase reproducibility of the measures of the level of a HIV disease marker, from assay-to-assay.
  • the "level" of a HIV disease prognosis marker is expressed as arbitrary units, since what is important is that the same kind of arbitrary units are compared (i) from assay-to-assay, or (ii) from one HIV-infected patient to others, or (iii) from assays performed at distinct time periods for the same patient, or (iv) between the HIV prognosis marker level measured in a patient's sample and a predetermined reference value (which may also be termed a "cut-off" value herein).
  • the measured value reflects the amount of anti-3S antibodies that is contained in the tested sample, or most preferably the concentration of anti-3S antibodies in the said sample.
  • the HIV viral load prognosis marker may be expressed in numerical units that reflect the number of copies of HIV genome contained in the patient's sample tested, or alternatively the concentration of HIV genome copies in the tested patient's sample.
  • HIV prognosis markers may be expressed in numerical units which reflect, respectively, (i) the number of CD4 T cells in the sample tested or alternatively the concentration thereof, and (ii) the percentage of CD4 T cells among the total lymphocyte cells present in the tested sample.
  • a sample collected from a patient encompasses any body fluid expected to contain antibodies and primarily in blood and bolld-derived substances.
  • a sample collected from a patient encompasses a whole blood sample, a serum sample and a blood plasma sample.
  • the in vitro method of the invention allows determining the likelyhood of an early or a delayed occurrence of an immunosuppression state in HIV-infected patients. Consequently, the in vitro method of prognosis of progression of an HIV disease of the invention may be practiced with the view of determining the period of time intervals for following up the clinical parameters of an HIV-infected patient and then for deciding of the opportunity to start an anti-retroviral therapeutic treatment.
  • the in vitro method of the invention allows determining the prognosis of progression of the HIV disease in HIV-infected patients that are already undergoing an immunosuppression state due to the viral infection.
  • the in vitro method of the invention allows discriminating between (i) HIV-infected immunosuppressed patients having a high likelihood to enter early into a symptomatic stage of the HIV disease and (ii) HIV-infected immunosuppressed patients having a low likelihood to enter early into a symptomatic stage of the HIV disease.
  • the examples herein show that the in vitro method for prognosis of progression of the HIV disease of the invention allows to determine between (i) HIV-infected patients having a high likelihood of a long term survival and (ii) HIV-infected patients having a low likelihood of a long term survival.
  • the level of anti-3S antibodies may be measured by any antibody detection method that is well known from the one skilled in the art.
  • the level of anti-3S antibodies may be measured by any kind of immunoassays including, but not limited to, ELISA, radioimmunoassays, fluorescence immunoassays, immunoaffinity chromatography, immuno-precipitation and the like.
  • the level of anti-3S antibodies is measured by an immunosorbent assay, which encompasses an ELISA method.
  • the level of the anti-3S antibodies is expressed as a value that refers to an actual signal that is generated, directly or indirectly, by the complexes formed between (i) 3S peptides used as baits in the antibody detection method and (ii) the anti-3S antibodies present in the patient's sample tested.
  • Arbitrary Units are generally determined after substracting the background noise signal, the latter being measured from a sample of the same kind than that of the patient's sample (e.g. a blood serum sample or a blood plasma sample) that is known to be exempt from anti-3S antibodies.
  • the Arbitrary Units are determined after calibration of the antibody detection method, using a serial of calibration standards (e.g. a serial of samples wherein each sample contains a known amount of anti-3S antibodies).
  • the quantification value of the level of anti-3S antibodies that is measured at step a) is compared to a predetermined reference value that is indicative of an HIV disease prognosis.
  • the said predetermined reference value is "indicative of an HIV disease prognosis” since it allows discrimination between (i) a "good” prognosis of HIV disease progression for values measured at step a) that are above the said reference value and (ii) a "bad" prognosis of HIV disease progression for values measured at step a) that are below the said reference value.
  • the inventors have determined an anti-3S antibody level value that allows to discriminate between (i) patients with a favourable outcome, including a delayed spontaneous HIV disease progression and a long survival time and (ii) patients with a poor outcome, including a non-delayed spontaneous HIV disease progression and a short survival time.
  • the predetermined reference value which may also be termed the "cut-off" value, is of 50.
  • the ELISA test format disclosed in the examples a sample collected from an HIV-infected patient where an anti-3S antibody level value of less than 50 has been measured at step a) of the in vitro method according to the invention is classified as a patient with a non-favourable outcome.
  • a sample collected from an HIV-infected patient where an anti-3S antibody level value of more than 50 has been measured at step a) of the in vitro method according to the invention is classified as a patient with a favourable outcome.
  • the reference value used for comparison at step b) of the method which may also be termed a "cut-off" value), may be determined as described hereunder.
  • the reference (“cut-off") value for the anti-3S antibodies prognosis marker may be predetermined by carrying out a method comprising the steps of:
  • the said method allows the setting of a "cut-off" value permitting discrimination between bad and good outcome prognosis.
  • a bad outcome prognosis encompasses a high likelihood that the onset of clinical symptoms of AIDS occurs within a 6-72 months time period following the time of infection with HIV, as well as a short time period of survival for patients having a CD4 T cell count below 500 cells/mm 3 (which includes patients having a CD4 T cell count below 200 cells/mm 3 )
  • a good outcome prognosis encompasses a low likelihood that the onset of clinical symptoms of AIDS occurs within a 6-72 months time period following the time of infection with HIV, as well as a long time period of survival for patients having a CD4 T cell count below 500 cells/mm 3 (which includes patients having a CD4 T cell count below 200 cells/mm 3 .
  • the said information relating to the actual clinical outcome of the HIV-infected patients are selected from the group consisting of (i) the duration of the AIDS-free survival (AFS) and (ii) survival time of patients having a CD4 T cell count below 500 CD4 cells/mm 3 or below 200 CD4 cells/mm 3 (TCDB).
  • the accuracy of the HIV disease prognosis when using the level of anti-3S antibodies as a prognosis marker, may be further improved by combining the said prognosis marker with one or more HIV prognosis markers known perse, e.g. the HIV viral load.
  • the HIV disease prognosis accuracy of the level of anti-3S antibodies is significantly higher than the prognosis accuracy of one of the other known HIV prognosis markers, namely the viral load.
  • the examples also show that the prognosis accuracy of the in vitro HIV disease prognosis method of the invention may be improved by combining (i) the viral load marker and (ii) the level of anti-3S antibodies marker.
  • the said method further comprises the steps of:
  • the viral load may be measured through any one of the conventional techniques that are well known from the one skilled in the art.
  • the viral load may be measured by using the method disclosed by Jennings et al. (2005, J Clin Microbiol, Vol. 43(12) : 4950-4956 ).
  • the reference value for the viral load quantification may be determined by using the same methods as those described herein for calculating the predetermined reference value (or "cut-off" value) of the level of anti-3S antibodies.
  • the predetermined reference value (or "cut-off" value) for the viral load marker may vary, depending of the viral load assay format which is used.
  • a predetermined reference value (cut-off) value of 4 log for the viral load marker has been used in the examples for performing the in vitro HIV disease prognosis method of the invention.
  • the in vitro HIV disease prognosis method of the invention may be performed by combining the anti-3S antibodies prognosis marker to one or more other HIV disease prognosis marker.
  • the said one or more HIV disease prognosis markers may be selected from the group consisting of (i) the HIV viral load, (ii) the absolute CD4 T cells count and (iii) the percentage of CD4+ T lymphocytes.
  • the in vitro method for the prognosis of progression of a HIV disease may comprise the steps of:
  • the said one or more other HIV disease prognosis markers are selected from the group consisting of (i) the HIV viral load, (ii) the absolute count of CD4 T cells, (iii) the percentage of CD4 T cells and (iv) the ratio of CD4+/CD8+ T lymphocytes. These include the following combinations of HIV disease prognosis markers:
  • a "good” prognosis of progression of the HIV disease or a "bad” prognosis of progression of the HIV disease is determined. Indeed, (i) a anti-3S antibody value above the predetermined value is classified as a "good” prognosis, whereas (ii) a viral load value above the predetermined reference value is classified as a "bad” prognosis parameter.
  • the results of step b) encompasses situations wherein (i) for at least one HIV prognosis marker tested, the marker value measured at step a) is classified as a "bad" prognosis for the said marker (i.e. the marker may be marked “Low”) and (ii) for at least one other HIV prognosis marker tested, the marker value measured at step a) is classified as a "good” prognosis for the said marker (i.e. the marker may be marked "High”).
  • the prognosis for progression of the HIV disease is determined by taking into account the number, and optionally the respective statistical weights, of "High” markers and of "Low” markers.
  • the measured value for each marker tested is balanced by affecting to the said measured marker value a numerical factor that materializes its weight of statistical significance in the prognosis of progression of the HIV disease.
  • step b) is performed by mathematically integrating the values measured at step a) for each HIV prognosis marker tested and their comparison to each corresponding reference values, so as to generate a single prediction value (e.g. "bad" prognosis or "good” prognosis) of the resulting composite HIV disease prognosis marker.
  • a single prediction value e.g. "bad" prognosis or "good” prognosis
  • a composite prognosis marker for HIV disease progression may result from a linear combination of each of the individual prognosis markers included therein, where logistic regression may be used to estimate the respective weight of each individual prognosis marker within the composite prognosis marker.
  • logistic regression may be used to estimate the respective weight of each individual prognosis marker within the composite prognosis marker.
  • an appropriate mathematical model could be used to establish the prognostic value of the used combination of markers.
  • the present invention further provides a method for monitoring the efficacy of a therapeutic treatment for HIV infection, by providing an accurate prognostic indicator of disease progression.
  • a monitoring method is very important both (i) for identifying possible therapeutic drugs and (ii) for monitoring patients undergoing therapies with these drugs.
  • the present invention deals with a method for monitoring the efficacy of a therapeutic treatment comprising the steps of:
  • the therapeutic treatment that is performed at step a) is continued if a "good" prognosis is determined, e.g. a high likelihood of a long survival time without entering into the AIDS stage.
  • the therapeutic treatment is adapted (e.g. is administered at a higher dosage or one or more of the active ingredients are replaced by more efficient ones) if a "bad" prognosis is determined, e.g. a low likelihood of a long survival time without entering into the AIDS stage.
  • the invention contemplates administration of an effective amount of an antiviral agent (or of a combination of antiviral agents) to an HIV-infected individual and monitoring the therapeutic outcome.
  • the in vitro method of prognosis for progression of a HIV disease is preferably performed by known immunodetection methods.
  • the examples herein illustrate embodiments of performing the in vitro HIV prognosis method of the invention by an enzyme-linked immunosorbent assay (ELISA), that is a highly conventional immunodetection method.
  • ELISA enzyme-linked immunosorbent assay
  • an ELISA may be performed either as a non-competitive immunoassay or as a competitive immunoassay.
  • unlabeled antigen e.g. a 3S peptide
  • a solid support or reaction vessel such as the surface of a microtiter plate or biochip.
  • the biological sample e.g. the sample collected from an HIV-infected patient
  • the antibodies present in the biological sample e.g. the anti-3S antibodies present in the sample collected from an HIV-infected patient
  • the immune complexes After the immune complexes have formed, excess biological sample is removed and the vessel is washed to remove non-specifically bound antibodies.
  • the immune complexes are then reacted with an appropriate enzyme-labeled anti-immunoglobulin (which may be also termed "secondary antibody").
  • the secondary antibody reacts with the antibodies (e.g. the anti-3S antibodies) in the immune complexes, not with other antigens bound to the reaction vessel.
  • Secondary antibodies specific for binding human antibodies are well known in the art and are commercially available, such as from Sigma Chemical Co. (St Louis, MO, USA).
  • the enzyme substrate is added.
  • the enzyme linked to the secondary antibody catalyzes a reaction that converts the substrate into a detectable product.
  • excess antigen is present, the amount of product is directly proportional to the amount of antibodies (also termed "primary antibodies") present in the patient's sample.
  • the product is fluorescent or luminescent, which can be measured using technology and equipment well known in the art.
  • the enzyme reaction results in the conversion of the enzyme substrate into a coloured product, which can be measures spectrophotometrically.
  • Typical enzymes that can be linked to secondary antibodies include horseradish peroxidise, glucose oxidase, glucose-6-phosphate dehydrogenase, alkaline phosphatise, beta-galactosidase and urease.
  • suitable solid supports include organic and inorganic polymers, e.g. dextrans, natural or modified celluloses, polyethylene, polystyrene, polyacrylamides, etc.
  • the said 3S peptide may be obtained either (i) by chemical synthesis or (ii) by recombinant expression, e.g. in E. coli cells transformed with an expression cassette (eventually inserted in a recombinant vector, i.e. a recombinant plasmid) encoding the said 3S peptide.
  • Example 1 Identification of the level of anti-3S antibodies as a prognosis marker of the progression of a HIV disease
  • the ANRS SEROCO/HEMOCO cohort started in 1988 has enrolled HIV-infected adults referred from 21 hospitals and a network of private practitioners. The study was approved by the Ethics Committee and all subjects gave written informed consent. Patients receive thorough clinical and laboratory examinations at inclusion and are then seen every 3 or 6 months according to their clinical status. All AIDS defining illnesses are checked in the medical files and reviewed with the participating physicians. The clinical status of patients lost to follow-up is assessed by crosschecking with the national AIDS register. Sera collected at enrolment and at each 6 month visit are stored at minus 180°C.
  • HIV seroconversion was documented by an interval of less than 24 months between a negative and a positive HIV antibody test, or an incomplete Western blot followed by a complete Western blot.
  • the date of infection was defined as the date of the incomplete Western blot minus 1 month, or the date of a primary symptomatic infection minus 15 days, or the midpoint between the two tests (Hubert 2000). 244 seroconverters with available frozen sera were included in this analysis.
  • follow-up was censored in 1996, before cART widespread use era. The median year of infection was 1989 and the median follow-up time since infection was 6.5 years.
  • the ELISA assay was performed on flat-bottom 96-well microplates (Maxisorp, Nunc).
  • the plates were coated with the 3S-peptide at 2 mg/ml in PBS, overnight 17 h at 4°C. Plates were washed twice in PBS/0,1%Tween, and then blocked in PBS/3% nonfat milk for 2 h at 37°C.
  • the tested serum/plasmas were heat-inactivated (56°C, 45 min) and then added at various concentrations (1/20, 1/200 and 1/2000) in triplicate for 1h30 min h at 37°C. Serums are diluted in assay buffer (PBS, 3% non fat milk, 0,1% tween-20).
  • biotin-SP-conjugated rabbit anti-human IgG Jackson ImmunoResearch
  • the chemical reaction is stopped with 4N H 2 SO 4 , and analyzed at 450 nm (Microplate reader, Molecular Devices).
  • Negative controls used human AB serum (SAB) and plasma from 10 uninfected donors diluted at 1/20 in assay buffer.
  • Quantification of anti-3S antibodies used calibration standards including dilutions of a purified mouse anti-3S monoclonal antibody, named 15C8f2.
  • the standard curve corresponds to serial 2-fold dilutions of this monoclonal antibody, with value comprised between 0 and 200 ng/ml.
  • the monoclonal antibody is diluted in assay buffer.
  • the OD of the tested samples are reported within the linear part of the standard curve to obtain the corresponding concentration of 15C8F2 monoclonal antibody giving the same signal.
  • the values of anti-3S antibodies are determined by taking into account the dilution factor of the tested samples and the volume.
  • the detection limit is set by the values obtained for the negative control and fixed to a value of 10 arbitrary units.
  • CD4 lymphocyte counts are determined at each visit by means of flow cytometry. All HIV-1 RNA levels were determined retrospectively from stored sera.
  • the three participating university labs used reverse transcriptase-polymerase chain reaction (Amplicor HIV-1 Monitor assay, Roche Molecular Systems, Neuilly-sur-Seine, quantification threshold 400 copies/mL). Early anti-3S antibodies levels were measured at enrolment from frozen sera (median time since estimated date of infection: 9 months).
  • the results of figures 1 and 2 show that the level of anti-3S antibodies consists of an actual prognosis marker for the progression of a HIV disease, and in particular for prognosis of survival in HIV-infected patients having a CD4 T cell count above 200/mm 3 .
  • Figure 3 illustrates an analysis of the level of HIV viral load for the first 36 months after enrolment, the viral load values being classified as above or below the cut-off value of 4 log.
  • Figure 4 illustrates the combined analysis of (i) the level of anti-3S antibodies (above (“Hi”) or below (“Lo”) the cut-off value of 50) and (ii) the level of the HIV viral load (above or below the cut-off value of 4 log), for 36 months subsequent to contamination.
  • Figure 6 illustrates prognosis of the risk of progression towards AIDS when using the combined measure of (i) the level of anti-3S antibodies and (ii) the level of the HIV viral load), for 120 months subsequent to contamination.
  • Table 2 also show that the risk of progression, over 36 month period, towards a CD4 T cell count of less than 200 CD4 T cells/mm 3 is delayed in patients with a level of anti-3S antibodies above the cut-off value of 50, after adjustment with age, sex, VIH RNA and DNA.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Organic Chemistry (AREA)
  • AIDS & HIV (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Claims (8)

  1. Méthode in vitro de pronostic de la progression d'une maladie associée à une infection par le VIH-1 chez un patient infecté avec un virus VIH-1, n'ayant pas le phénotype rare de survie des asymptomatiques à long terme, lequel pronostic est basé sur le taux d'anticorps dirigés contre le peptide 3S de SEQ ID N° 2 en tant que marqueur de pronostic, lequel marqueur de pronostic est indépendant du marqueur de pronostic de nombres de CD4, ladite méthode comprenant les étapes consistant à :
    a) mesurer le taux d'anticorps dirigés contre le peptide 3S de SEQ ID N° 2 dans un échantillon recueilli auprès dudit patient,
    b) comparer le taux d'anticorps anti-3S mesuré à l'étape a) à une valeur de référence du taux d'anticorps anti-3S révélatrice de la progression de la maladie associée à une infection par le VIH-1.
  2. Méthode selon la revendication 1, dans laquelle l'étape a) est réalisée par immunodétection des anticorps dirigés contre le peptide de SEQ ID N° 2.
  3. Méthode selon la revendication 1, dans laquelle l'étape a) est réalisée par dosage ELISA en utilisant un polypeptide comprenant le peptide de SEQ ID N° 2 immobilisé sur un support solide.
  4. Méthode selon la revendication 1, définie en outre comme comprenant les étapes suivantes consistant à :
    1) mesurer la charge virale du VIH-1 dans ledit échantillon recueilli auprès dudit patient, et
    2) comparer la valeur de la charge virale mesurée à l'étape 1) avec une valeur de référence révélatrice de la progression de la maladie associée à une infection par le VIH-1.
  5. Méthode in vitro de pronostic de la progression d'une maladie associée à une infection par le VIH-1 selon la revendication 1, dans laquelle :
    - l'étape a) comprend en outre la mesure d'au moins un autre marqueur de pronostic de la maladie associée à une infection par le VIH-1 dans un échantillon recueilli auprès d'un patient infecté avec le VIH-1, et
    - l'étape b) comprend la comparaison, pour chaque marqueur de pronostic de la maladie associée à une infection par le VIH-1 mesuré à l'étape a), de la valeur résultante du marqueur avec une valeur de référence pour ledit marqueur de pronostic.
  6. Méthode in vitro selon la revendication 5, dans laquelle ledit ou lesdits autres marqueurs de pronostic de la maladie associée à une infection par le VIH-1 sont choisis dans le groupe constitué par (i) la charge virale du VIH, (ii) le nombre absolu de lymphocytes T CD4, (iii) le pourcentage de lymphocytes T CD4 et (iv) le rapport de lymphocytes T CD4+/CD8+.
  7. Méthode in vitro selon la revendication 6, dans laquelle l'étape a) comprend la mesure d'une combinaison de marqueurs de pronostic de la maladie associée à une infection par le VIH-1 choisis dans le groupe constitué par :
    - le taux d'anticorps anti-3S combiné avec le marqueur (i) ci-dessus,
    - le taux d'anticorps anti-3S combiné avec le marqueur (ii) ci-dessus,
    - le taux d'anticorps anti-3S combiné avec le marqueur (iii) ci-dessus,
    - le taux d'anticorps anti-3S combiné avec le marqueur (iv) ci-dessus,
    - le taux d'anticorps anti-3S combiné avec les marqueurs (i) et (ii) ci-dessus,
    - le taux d'anticorps anti-3S combiné avec les marqueurs (i) et (iii) ci-dessus,
    - le taux d'anticorps anti-3S combiné avec les marqueurs (i) et (iv) ci-dessus,
    - le taux d'anticorps anti-3S combiné avec les marqueurs (ii) et (iii) ci-dessus,
    - le taux d'anticorps anti-3S combiné avec les marqueurs (ii) et (iv) ci-dessus,
    - le taux d'anticorps anti-3S combiné avec les marqueurs (iii) et (iv) ci-dessus,
    - le taux d'anticorps anti-3S combiné avec les marqueurs (i), (ii) et (iii) ci-dessus,
    - le taux d'anticorps anti-3S combiné avec les marqueurs (i), (ii) et (iv) ci-dessus,
    - le taux d'anticorps anti-3S combiné avec les marqueurs (ii), (iii) et (iv) ci-dessus,
    - le taux d'anticorps anti-3S combiné avec les marqueurs (i), (iii) et (iv) ci-dessus, et
    - le taux d'anticorps anti-3S combiné avec les marqueurs (i), (ii), (iii) et (iv) ci-dessus.
  8. Méthode de surveillance de l'efficacité d'un traitement thérapeutique comprenant l'étape consistant à réaliser la méthode in vitro pour le pronostic de la progression d'une maladie associée à une infection par le VIH-1 selon l'une quelconque des revendications 1 à 7 sur un échantillon recueilli auprès d'un patient infecté avec le VIH-1 qui n'a pas le phénotype rare de survie des asymptomatiques à long terme et qui reçoit un traitement thérapeutique avec une composition pharmaceutique comprenant un ou plusieurs agents antirétroviraux.
EP12707649.5A 2011-02-22 2012-02-21 Méthode permettant de pronostiquer l'évolution de l'infection par le vih Not-in-force EP2678683B1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
PL12707649T PL2678683T3 (pl) 2011-02-22 2012-02-21 Sposób prognozowania progresji choroby wywołanej HIV
EP12707649.5A EP2678683B1 (fr) 2011-02-22 2012-02-21 Méthode permettant de pronostiquer l'évolution de l'infection par le vih
RS20160990A RS55354B1 (sr) 2011-02-22 2012-02-21 Postupak za prognozu napredovanja hiv-a

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP11305187 2011-02-22
PCT/IB2012/050787 WO2012114272A1 (fr) 2011-02-22 2012-02-21 Méthode permettant de pronostiquer l'évolution de l'infection par le vih
EP12707649.5A EP2678683B1 (fr) 2011-02-22 2012-02-21 Méthode permettant de pronostiquer l'évolution de l'infection par le vih

Publications (2)

Publication Number Publication Date
EP2678683A1 EP2678683A1 (fr) 2014-01-01
EP2678683B1 true EP2678683B1 (fr) 2016-08-17

Family

ID=44169279

Family Applications (1)

Application Number Title Priority Date Filing Date
EP12707649.5A Not-in-force EP2678683B1 (fr) 2011-02-22 2012-02-21 Méthode permettant de pronostiquer l'évolution de l'infection par le vih

Country Status (13)

Country Link
US (1) US20140024018A1 (fr)
EP (1) EP2678683B1 (fr)
JP (1) JP5952313B2 (fr)
CN (1) CN103460047B (fr)
CA (1) CA2827511A1 (fr)
DK (1) DK2678683T3 (fr)
ES (1) ES2603639T3 (fr)
HU (1) HUE030649T2 (fr)
PL (1) PL2678683T3 (fr)
PT (1) PT2678683T (fr)
RS (1) RS55354B1 (fr)
RU (1) RU2601379C2 (fr)
WO (1) WO2012114272A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2778681A1 (fr) * 2013-03-15 2014-09-17 Biomay Ag Procédé pour diagnostiquer une infection virale
RU2597805C1 (ru) * 2015-06-16 2016-09-20 Сергей Борисович Пономарёв Способ прогнозирования развития спида у вич-носителей
RU2697392C1 (ru) * 2019-03-12 2019-08-14 Наталья Юрьевна Пшеничная Способ прогноза прогрессирования ВИЧ-инфекции у больных, получающих антиретровирусную терапию
CN112562867A (zh) * 2021-02-22 2021-03-26 天津迈德新医药科技有限公司 一种预测极早期hiv感染风险的装置、存储介质和电子装置

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8030097B2 (en) * 2008-04-30 2011-10-04 Versitech Limited and R & C Biogenius Limited Lipocalin-2 as a prognostic and diagnostic marker for heart and stroke risks
WO2010040853A1 (fr) * 2008-10-10 2010-04-15 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédé pour le criblage de substances candidates actives contre l'infection d'un sujet par un virus du vih et kits pour mettre en œuvre ledit procédé

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "RESEARCH & DEVELOPMENT PROJECTS", INNAVIRVAX, 12 September 2009 (2009-09-12), pages 1, XP055208144, Retrieved from the Internet <URL:http://web.archive.org/web/20090912041108/http://www.innavirvax.fr/RD-projects/menu-id-4.html> [retrieved on 20150817] *
CASADO CONCEPCION ET AL: "Host and Viral Genetic Correlates of Clinical Definitions of HIV-1 Disease Progression", PLOS ONE, vol. 5, no. 6, E11079, June 2010 (2010-06-01), ISSN: 1932-6203 *
VAN DER HELM JANNIE J ET AL: "Characterisation of long-term non-progression of HIV-1 infection after seroconversion: a cohort study.", THE LANCET. HIV OCT 2014, vol. 1, no. 1, October 2014 (2014-10-01), pages e41 - e48, ISSN: 2352-3018 *
VIEILLARD VINCENT ET AL: "NK cytotoxicity against CD4+ T cells during HIV-1 infection: A gp41 peptide induces the expression of an NKp44 ligand", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 102, no. 31, 2 August 2005 (2005-08-02), pages 10981 - 10986, XP002341851, ISSN: 0027-8424, DOI: 10.1073/PNAS.0504315102 *

Also Published As

Publication number Publication date
CN103460047B (zh) 2016-06-15
JP2014507661A (ja) 2014-03-27
US20140024018A1 (en) 2014-01-23
PT2678683T (pt) 2016-11-24
PL2678683T3 (pl) 2017-08-31
CN103460047A (zh) 2013-12-18
ES2603639T3 (es) 2017-02-28
EP2678683A1 (fr) 2014-01-01
CA2827511A1 (fr) 2012-08-30
RU2601379C2 (ru) 2016-11-10
RS55354B1 (sr) 2017-03-31
WO2012114272A1 (fr) 2012-08-30
RU2013138260A (ru) 2015-03-27
DK2678683T3 (en) 2016-12-19
JP5952313B2 (ja) 2016-07-13
HUE030649T2 (en) 2017-05-29

Similar Documents

Publication Publication Date Title
Schwartz et al. Human immunodeficiency virus test evaluation, performance, and use: Proposals to make good tests better
Yang et al. A retrospective study of the C-reactive protein to lymphocyte ratio and disease severity in 108 patients with early COVID-19 pneumonia from January to March 2020 in Wuhan, China
Massanella et al. Long-term effects of early antiretroviral initiation on HIV reservoir markers: a longitudinal analysis of the MERLIN clinical study
Heide et al. Broadly directed SARS-CoV-2-specific CD4+ T cell response includes frequently detected peptide specificities within the membrane and nucleoprotein in patients with acute and resolved COVID-19
KR20140038440A (ko) Pml의 위험을 판단하는 방법
EP2678683B1 (fr) Méthode permettant de pronostiquer l&#39;évolution de l&#39;infection par le vih
Noh et al. Longitudinal assessment of anti-SARS-CoV-2 immune responses for six months based on the clinical severity of COVID-19
Spijkerman et al. Early and late HIV-1 RNA level and its association with other markers and disease progression in long-term AIDS-free homosexual men
Ivison et al. Natural killer cell receptors and ligands are associated with markers of HIV-1 persistence in chronically infected ART suppressed patients
Sangwan et al. Immune activation markers in individuals with HIV-1 disease and their correlation with HIV-1 RNA levels in individuals on antiretroviral therapy
Schüpbach et al. Diagnostic performance of line-immunoassay based algorithms for incident HIV-1 infection
Tiba et al. Immune reconstitution during the first year of antiretroviral therapy of HIV-1-infected adults in rural Burkina Faso
Shetty et al. Laboratory Tests for HIV: Diagnosing, monitoring and managing AIDS-an overview
DeSimone et al. New methods for the detection of HIV
Singh et al. IL-15 and sMAdCAM: Novel roles in COVID-19 pathogenesis
US20130244965A1 (en) Assays and methods for determining risk of a macrophage-mediated disease development in a subject infected with hiv
Sultana et al. Spectrum of thyroid dysfunctions among hospitalized patients with non-critically ill coronavirus disease 2019: A cross-sectional study
Matsunaga et al. Long-term follow-up of production of IgM and IgG antibodies against SARS-CoV-2 among patients with COVID-19
EP1123419B1 (fr) Méthode pour déterminer le pronostic d&#39;individus infectés par le vih
Elsayed A COVID-19 from Laboratory Suspicion to Confirmatory Tests: Controversial Debatable
Santos et al. COVID-19 recurrence is related to disease-early profile T cells while detection of anti-S1 IgG is related to multifunctional T cells
Rugeles López et al. Identification of CD8+ T cell subsets that normalize in early-treated people living with HIV receiving antiretroviral therapy
Indrati et al. The Protection Level of S-RBD SARS-CoV-2 Immunoglobulin G Antibodies Using The Chemiluminescent Immunoassay Compared To The Surrogate Virus Neutralization Test Method
Grant-McAuley Early Viral Suppression and the Antibody Response to HIV Infection in HPTN 071 (PopART)
Ciurea et al. STUDY ON THE INCIDENCE OF ANTI-SARS COV2 ANTIBODIES TO HEALTHCARE STAFF.

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20130923

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20140901

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

INTG Intention to grant announced

Effective date: 20160329

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 821575

Country of ref document: AT

Kind code of ref document: T

Effective date: 20160915

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602012021817

Country of ref document: DE

REG Reference to a national code

Ref country code: RO

Ref legal event code: EPE

REG Reference to a national code

Ref country code: NL

Ref legal event code: FP

REG Reference to a national code

Ref country code: PT

Ref legal event code: SC4A

Ref document number: 2678683

Country of ref document: PT

Date of ref document: 20161124

Kind code of ref document: T

Free format text: AVAILABILITY OF NATIONAL TRANSLATION

Effective date: 20161117

REG Reference to a national code

Ref country code: SE

Ref legal event code: TRGR

REG Reference to a national code

Ref country code: DK

Ref legal event code: T3

Effective date: 20161205

REG Reference to a national code

Ref country code: NO

Ref legal event code: T2

Effective date: 20160817

Ref country code: LT

Ref legal event code: MG4D

REG Reference to a national code

Ref country code: EE

Ref legal event code: FG4A

Ref document number: E012904

Country of ref document: EE

Effective date: 20161114

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160817

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160817

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 6

REG Reference to a national code

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2603639

Country of ref document: ES

Kind code of ref document: T3

Effective date: 20170228

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NL

Payment date: 20170123

Year of fee payment: 6

REG Reference to a national code

Ref country code: GR

Ref legal event code: EP

Ref document number: 20160402863

Country of ref document: GR

Effective date: 20170222

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: SE

Payment date: 20170124

Year of fee payment: 6

Ref country code: MC

Payment date: 20170124

Year of fee payment: 6

Ref country code: EE

Payment date: 20170119

Year of fee payment: 6

Ref country code: NO

Payment date: 20170124

Year of fee payment: 6

Ref country code: CH

Payment date: 20170125

Year of fee payment: 6

Ref country code: RO

Payment date: 20170201

Year of fee payment: 6

Ref country code: GR

Payment date: 20170124

Year of fee payment: 6

Ref country code: DE

Payment date: 20170119

Year of fee payment: 6

Ref country code: FR

Payment date: 20170227

Year of fee payment: 6

Ref country code: FI

Payment date: 20170120

Year of fee payment: 6

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602012021817

Country of ref document: DE

REG Reference to a national code

Ref country code: HU

Ref legal event code: AG4A

Ref document number: E030649

Country of ref document: HU

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160817

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160817

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160817

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: AT

Payment date: 20170120

Year of fee payment: 6

Ref country code: IE

Payment date: 20170123

Year of fee payment: 6

Ref country code: BG

Payment date: 20170120

Year of fee payment: 6

Ref country code: BE

Payment date: 20170119

Year of fee payment: 6

Ref country code: HU

Payment date: 20170131

Year of fee payment: 6

Ref country code: LU

Payment date: 20170119

Year of fee payment: 6

Ref country code: DK

Payment date: 20170120

Year of fee payment: 6

Ref country code: PT

Payment date: 20170201

Year of fee payment: 6

Ref country code: RS

Payment date: 20170124

Year of fee payment: 6

Ref country code: GB

Payment date: 20170124

Year of fee payment: 6

Ref country code: LV

Payment date: 20170119

Year of fee payment: 6

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: ES

Payment date: 20170123

Year of fee payment: 6

Ref country code: TR

Payment date: 20170214

Year of fee payment: 6

Ref country code: IT

Payment date: 20170120

Year of fee payment: 6

26N No opposition filed

Effective date: 20170518

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160817

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: PL

Payment date: 20170123

Year of fee payment: 6

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 602012021817

Country of ref document: DE

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: EE

Ref legal event code: MM4A

Ref document number: E012904

Country of ref document: EE

Effective date: 20180228

REG Reference to a national code

Ref country code: DK

Ref legal event code: EBP

Effective date: 20180228

Ref country code: NO

Ref legal event code: MMEP

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180228

Ref country code: MT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20170221

REG Reference to a national code

Ref country code: SE

Ref legal event code: EUG

REG Reference to a national code

Ref country code: NL

Ref legal event code: MM

Effective date: 20180301

REG Reference to a national code

Ref country code: AT

Ref legal event code: MM01

Ref document number: 821575

Country of ref document: AT

Kind code of ref document: T

Effective date: 20180221

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20180221

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180221

Ref country code: RO

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180221

Ref country code: PT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180821

Ref country code: AL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160817

Ref country code: EE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180228

Ref country code: NO

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180228

Ref country code: HU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180222

Ref country code: SE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180222

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

REG Reference to a national code

Ref country code: BE

Ref legal event code: FP

Effective date: 20161117

Ref country code: BE

Ref legal event code: MM

Effective date: 20180228

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180228

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180228

Ref country code: LV

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180221

Ref country code: RS

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180829

Ref country code: AT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180221

Ref country code: GR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180904

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180221

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

Effective date: 20181031

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180301

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180901

Ref country code: DK

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180228

Ref country code: BG

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180903

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180221

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180228

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180228

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180221

Ref country code: IT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180221

REG Reference to a national code

Ref country code: ES

Ref legal event code: FD2A

Effective date: 20190801

REG Reference to a national code

Ref country code: AT

Ref legal event code: UEP

Ref document number: 821575

Country of ref document: AT

Kind code of ref document: T

Effective date: 20160817

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180222

Ref country code: CY

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160817

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160817

Ref country code: PL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180221

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20161217

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180221